Evaluation of different sampling methods and results of real-time PCR for detection of feline herpes virus-1, Chlamydophila felis and Mycoplasma felis in cats

Objective To investigate how different sampling techniques affect detection of DNA from feline herpes virus Type 1 (FHV-1), Chlamydophila felis and Mycoplasma felis and to study the correlation between positive test results and clinical signs in cats. Animals Fifty-one cats; 24 with ocular signs and 27 healthy control cats. Procedures Samples were collected from all cats using cotton swabs, conjunctival and corneal biopsies, and corneal scrapings. Samples were analyzed for presence of FHV-1, C. felis, M. felis, and feline DNA, defined by 28S rDNA, by using real-time PCR. Results In affected cats, FHV-1 was detected in only one cat; C. felis and M. felis were not detected in any affected cats. None of the three organisms was detected in any control cats. Feline DNA was demonstrated in all conjunctival samples, in 82% of corneal swabs, 92% of corneal scrapings, and 100% of keratectomy samples. Conclusions Because of the generally low detection rate for FHV-1, C. felis, and M. felis DNA in this study, differences regarding sampling technique could not be determined and correlation between positive test results and degree of clinical signs could not be made. Detection of feline DNA in most samples irrespective of sampling technique, suggests a low prevalence of FHV-1, C. felis and M. felis in this population of cats.     

Cytologic findings, and feline herpesvirus DNA and Chlamydophila felis antigen detection rates in normal cats and cats with conjunctival and corneal lesions

Samples were collected from 36 cats with feline herpesvirus (FHV-1)-related ocular disease (conjunctivitis, epithelial or stromal keratitis, or corneal sequestration), and 17 cats without ocular changes. Corneoconjunctival swabs, scrapings and biopsies were tested in various combinations for presence of FHV-1 DNA using single round (sr) polymerase chain reaction (PCR) and nested PCR (nPCR). Additional swabs from the inferior conjunctival fornix were tested by enzyme-linked immunosorbent assay for Chlamydophila felis antigen. Cytologic evaluation was carried out on conjunctival (cats with conjunctivitis) and corneal (cats with keratitis) cytobrush preparations. FHV-1 DNA was detected by PCR in 14 (39%) cats with ocular disease and 1 (6%) of the control group. Agreement between srPCR and nPCR results was significant (P < 0.01). FHV-1 DNA was detected in 3/7 cats with conjunctivitis, 5/6 cats with epithelial keratitis, 3/11 cats with stromal keratitis, and 3/12 cats with corneal sequestration. There was a significant association (P = 0.0027) between viral presence and epithelial keratitis. However, no significant association was found between viral presence and conjunctivitis (P = 0.059), stromal keratitis (P = 0.15), or corneal sequestration (P = 0.18). With respect to FHV-1 DNA detection, intersample agreement was significant (P < 0.03). No sampling technique seemed more likely than another to harvest detectable viral DNA, except for cats with corneal sequestrum in which viral DNA was not detected using corneoconjunctival swabs. FHV-1 DNA was detected in 6/9 samples with intranuclear inclusion bodies and in 6/7 cats with eosinophils on cytologic examination. All samples tested negative for C. felis antigen.     

Prevalence of Chlamydophila felis and feline herpesvirus 1 in cats with conjunctivitis in northern Italy

The prevalence of Chlamydophila felis and feline herpesvirus 1 (FHV-1) infection in cats with conjunctivitis in northern Italy was investigated by conventional polymerase chain reaction (PCR) testing. In cats with conjunctivitis, C felis and FHV-1 were detected in 14 of 70 (20%) and in 23 of 70 (33%) animals, respectively. None of the 35 control cats were positive for C felis, whereas 7 (20%) of these cats were positive for FHV-1. Mixed infections were present in 5 of 70 cats (7%). Cats positive for C felis were significantly younger than control animals (P = .02), whereas no significant age differences were observed between FHV-1-positive cats and control cats (P = .41) or between FHV-1-positive animals and C felis-positive animals (P = .16). Cats sampled during acute-phase conjunctivitis were also investigated for the presence of C felis by conjunctival scrapings. In this acute phase, substantial agreement was found when comparing the results of the 2 methods (K = .80). The association between PCR results and conjunctivitis was evaluated for the 2 pathogens. The presence of C felis was significantly associated with conjunctivitis (P = .004), whereas the detection of FHV-1 did not significantly correlate with the clinical sign (P = .25), suggesting that, by itself. PCR is not suitable for the diagnosis of FHV-1-related conjunctivitis.     

Update on pathogenesis, diagnosis, and treatment of feline herpesvirus type 1

Feline herpesvirus type 1 (FHV-1) infection, but not necessarily chronic or recurrent disease, is common throughout domestic cat populations worldwide. Knowledge of a few essential virological facts permits practitioners to provide appropriate advice to owners of individual pet cats infected with this virus and to assist in the management of shelters and other multicat households in which the virus is enzootic. This article discusses pathogenesis, diagnostic techniques, and clinical signs considered characteristic of infection with FHV-1. Treatment options are considered under the broad categories of supportive care, antiviral agents, and adjunctive therapies.     

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.     

Quantitative real-time PCR assays for the concurrent diagnosis of infectious laryngotracheitis virus,Newcastle disease virus and avian metapneumovirus in poultry

Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log₁₀ dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%–98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.     

The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R² = 0.93, slope = −3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.     

Effect of carryover and presampling procedures on the results of real-time PCR used for diagnosis of bovine intramammary infections with Streptococcus agalactiae at routine milk recordings

The use of PCR tests as diagnostics for intramammary infections (IMI) based on composite milk samples collected in a non-sterile manner at milk recordings is increasing. Carryover of sample material between cows and non-aseptic PCR sampling may be incriminated for misclassification of IMI with Streptococcus agalactiae (S. agalactiae) in dairy herds with conventional milking parlours. Misclassification may result in unnecessary costs for treatment and culling. The objectives of this study were to (1) determine the effect of carryover on PCR-positivity for S. agalactiae at different PCR cycle threshold (Ct) cut-offs by estimating the between-cow correlation while accounting for the milking order, and (2) evaluate the effect of aseptic presampling procedures (PSP) on PCR-positivity at the different Ct-value cut-offs.     

In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.     

地塞米松、油酸、乳酸对体外培养脂肪细胞HSL mRNA表达的影响

为了研究地塞米松、油酸、乳酸对HSL mRNA表达的影响,试验采用实时荧光定量方法检测脂肪细胞中HSL mRNA的表达量。结果表明:油酸、乳酸抑制脂肪细胞内HSL mRNA表达,抑制作用随着浓度增加而增强;地塞米松促进脂肪细胞内HSL mRNA表达,促进作用存在剂量依赖性。     

ACVIM consensus statement guidelines for the classification,diagnosis, and management of cardiomyopathies in cats

Cardiomyopathies are a heterogeneous group of myocardial disorders of mostly unknown etiology, and they occur commonly in cats. In some cats, they are well-tolerated and are associated with normal life expectancy, but in other cats they can result in congestive heart failure, arterial thromboembolism or sudden death. Cardiomyopathy classification in cats can be challenging, and in this consensus statement we outline a classification system based on cardiac structure and function (phenotype). We also introduce a staging system for cardiomyopathy that includes subdivision of cats with subclinical cardiomyopathy into those at low risk of life-threatening complications and those at higher risk. Based on the available literature, we offer recommendations for the approach to diagnosis and staging of cardiomyopathies, as well as for management at each stage.     

氯化胆碱、肌醇和胆汁酸对鲤鱼生长、血清生化及免疫指标的影响

在基础饲料中分别添加氯化胆碱、肌醇和胆汁酸,以基础饲料为对照组饲喂鲤鱼,观察抗应激添加剂对鲤鱼生长性能、血清生化指标和血清非特异性免疫指标的影响。经过60d的饲喂试验,结果表明:①3种物质的添加均有良好的促生长效果,其中氯化胆碱组、肌醇组和胆汁酸组的增重率与对照组相比显著提高(P<0.05),饲料系数与对照组相比显著降低(P<0.05);②3种物质中,肌醇和胆汁酸的添加对提高鲤鱼的免疫力作用更明显,其中肌醇能显著提高血清碱性磷酸酶(ALP)、溶菌酶(LSZ)和超氧化物歧化酶(SOD)的活性(P<0.05),显著降低血清天门冬氨酸转氨酶(AST)的活性(P<0.05);胆汁酸的添加可以显著提高溶菌酶(LSZ)的活性(P<0.05),显著降低丙氨酸转氨酶(ALT)和天门冬氨酸转氨酶(AST)的活性(P<0.05);氯化胆碱的添加可以显著降低鲤鱼血清丙氨酸转氨酶(ALT)活性(P<0.05)。     

探针实时荧光定量PCR检测白藜芦醇体外抗鸭瘟病毒活性方法的建立及应用

建立以TaqMan荧光探针为特点的PCR,检测白藜芦醇体外抗鸭瘟病毒活性。本研究针对鸭瘟病毒UL30基因序列设计特异性引物和TaqMan荧光探针,建立鸭瘟病毒TaqMan探针实时荧光定量PCR检测方法,并验证方法的特异度、敏感度和稳定性及对临床样品进行检测;运用该方法检测白藜芦醇对鸭胚成纤维细胞接种鸭瘟病毒后细胞中病毒增殖情况的影响,用以评价该药物体外抗鸭瘟病毒效果。结果显示,该方法建立的定量标准曲线阈值循环数（Threshold cycle,Ct）与模板拷贝数呈良好线性关系（R2=0.997）,斜率为-3.328,扩增效率（E）为99.7%,具有良好的特异度、敏感度和稳定性并能对临床样本进行准确的检测。运用该方法检测白藜芦醇体外抗鸭瘟病毒结果显示：经白藜芦醇处理后,染毒细胞中病毒拷贝数为（7.71±0.37）×10^4,与病毒对照组比较（5.63±0.26）×10^6明显减少（P〈0.01）。结果表明,TaqMan探针实时荧光定量PCR方法建立并成功用于体外药物抗鸭瘟病毒的检测;白藜芦醇具有良好的体外抗鸭瘟病毒活性。     

Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens

A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122 bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.     

Recovery from adverse effects of heat stress on slow-growing chicks in the tropics 1: Effect of ascorbic acid and different levels of betaine

Three hundreds, 21 d-old slow-growing chicks were randomly divided among 5 treatments, of 5 replicates each. Each replicate contained 12 unsexed chicks housed in (1 × 1) a floor pen. A group was kept under thermoneutral condition at 28 ± 4°C and RH was 55 ± 3% during 21–84 d of age (positive control) and fed corn-soybean meal diet. The other four groups were kept for three successive days per week under heat stress (HS) at 38 ± 1.4°C and 49 ± 2% RH from 12.00 to 16.00 pm. Chicks in HS treatments were fed corn-soybean meal diet without (negative control) or with 250 mg AA/kg diet and Bet at 0.5 and 1 g/kg diet. HS decreased productive performance, increased (P < 0.05) meat dry matter, plasma triglyceride and serum calcium whereas decreased (P > 0.05) plasma glucose, serum total protein and water holding capacity (WHC) of meat. AA and 1 g of Bet/kg diet was equally potent for partial relief (P < 0.05) of the negative effect of HS on growth, increased (P < 0.05) feed intake, protein digestibility (P < 0.05), dressing out percentage, liver and giblets, whilst improved (P < 0.05) feed conversion ratio (FCR). Also, a complete recovery from the negative effect (P < 0.05) of HS shown on plasma glucose and partial recovery (P < 0.05) observed in total protein, triglyceride, blood pH, packed cell volume (PCV), hemoglobin (Hgb), rectal temperature (RT) and respiration rate (RR) and improved humoral immune competence to sheep red blood cell (SBRCs) test.